Parkinson's Disease is a chronic central nervous system disorder characterized by a disturbance of voluntary movement in which muscles become stiff and sluggish, movement becomes clumsy and difficult, and uncontrollable rhythmic twitching of groups of muscles produces characteristic shaking or tremor. At the present time, approximately 1.5 million Americans are diagnosed with Parkinson's Disease. The condition of Parkinson's Disease is believed to be caused by a degeneration of pre-synaptic dopaminergic neurons in the central nervous system, possibly in the substantia nigra region of the brain, due to absence of adequate release of a chemical transmitter, dopamine (“DA”), during neuronal activity. DA is partly responsible for starting a circuit of messages that coordinate normal movement of the body.
In the neurons, DA is produced by decarboxylation of 3,4-dihydroxyphenylalanine (“DOPA”), the immediate precursor of dopamine (DA). DOPA in turn is produced by hydroxylation of tyrosine. The enzyme tyrosine hydroxylase (TH) catalyzes the rate-limiting step of converting tyrosine to DOPA. The activity of TH in the peripheral and central nervous system is subject to short and long term regulation by extra- and intracellular signals. Such signalling provides the mechanisms for modulation of the amount of DA (as well as norepinephrine and epinephrine) synthesized and available for secretion in response to physiological requirements.
Recent studies, which were based on examination of postmortem brains from Parkinson's Disease patients and animal models, have suggested that at least a part of the neuron loss is due to apoptosis, a genetically programmed cell suicidal program. (Anglade et al. Histol. Histopathol. 1997; 12(1):25-31).
Apoptosis, or programmed cell death, is a principal mechanism by which organisms eliminate unwanted cells. Normal tissues in the body are formed either by cells that have reached a terminally differentiated state and no longer divide or by cells that die after a period of time and are replaced from a pool of dividing cells. For example, nervous tissue is formed early in development and the cells of the nervous system reach a terminally differentiated state soon after birth. When nervous tissue is damaged, the nerve cells are incapable of dividing and, therefore, the loss of function due to the damaged nerve cells is not repaired. The deregulation of apoptosis, either excessive apoptosis or the failure to undergo it, has been implicated in a number of diseases such as cancer, acute inflammatory and autoimmune disorders, ischemic diseases and certain neurodegenerative disorders, including Parkinson's Disease.
During apoptosis, a range of cellular molecules are inappropriately activated and subsequently destroy the cell. In mammals, the central execution molecules for apoptosis are a group of enzyme called caspases. Among the 14 caspases identified so far, caspase 3 appears to play a central role in the final execution of cell death. Upon activation, caspase 3 cleaves a variety of essential cytoplasmic and nuclear proteins, ensuring the inevitability of cell death. (Dodel et al. Brain Res. Mol. Brain Res. 1999; 64(1):141-148; Schierle et al. Nat. Med. 1999; 5(1):97-100; Hartmann et al. PNAS 2000; 97(6):2875-2880; Anantharam et al. J. Neurosci. 2002; 22(5):1738-1751).
As a result of the dopaminergic neuron death, the neurons in the next part of basal ganglia called striatum are not adequately stimulated, resulting in the symptoms of PD. At the cellular level, round protein aggregates called Lewy bodies are seen in the damaged and dying neurons of the substantia nigra. The first symptoms of PD do not appear until there are substantial loss (about 50%) of neurons in the substantia nigra and more than 80% reduction in dopamine levels. The classic trio of primary symptoms of PD is tremor at rest, rigidity (stiffness) and bradykinesia (slow movement).
Currently, there is no curative treatment for PD. There is no medication that slows or stops the progression of PD either. Currently available medications for PD are symptomatic therapies that suppress or reduce the symptoms of PD.
Levodopa is the most effective treatment currently available for the symptoms of PD. Most PD patients will eventually be on levodopa treatment. Carbidopa is frequently given together with levodopa to prevent its conversion in the intestine and blood, reducing the side effects of levodopa (nausea and vomiting) and increasing the amounts of levodopa available to the brain. However, after years of levodopa treatment, there may be a “wearing-off” of its beneficial effects. PD patients may experience “end of dose failure”, that is, patients may feel good (“on”) for a period of time and then the PD symptoms return (“off”) before the next dose is taken. This on-off phenomenon is difficult to treat. In addition, patients may experience dyskinesias at an overdosage or peak-dose of levodopa. That is, patients may have abnormal involuntary movements with irregular, flowing, dance-like or jerky motion in any or all parts of the body.
Other symptomatic treatments of PD includes anticholinergics (e.g., trihexyphenidyl, benztropine, and biperiden), monoamine oxidase inhibitor (MOI, e.g., selegiline), dopamine receptor agonists (e.g., pergolide, bromocriptine, pramipexole and ropinirole), amantadine, catechol-o-methyltransferase (COMT) inhibitors (e.g., tolcapone and entacapone), and less frequently, some beta-blockers. Uses of these symptomatic agents involve side effects such as dry mouth, decreases memory, confusion, blurred vision, difficulty with urination, worsening constipation, sleep problems, nausea, nightmares, hallucinations, benign skin discoloration, liver damage (i.e., tolcapone), low blood pressure, slow heart rate and depression, etc.
In short, there remains the great demand for a medication that stops or slows the progression of PD while in the meantime causes fewer side effects.
In investigating PD and searching for a treatment, toxin-induced and genetic experimental models have been invaluable. The most established method is the treatment of animals with toxins that are specific for dopamine neurons, e.g., 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). After injection into an animal, these toxins are transported into dopaminergic neurons by selective uptake systems. These toxins concentrate in the neurons and eventually cause cell death, reproducing many characteristic features of PD. Both 6-OHDA and MPTP can replicate the neurochemical, morphologic, and behavioral changes seen in human disease (Tolwani et al. Laboratory Animal Science 1999; 49(4):363-371). These animal PD models are useful for studying many aspects of the PD and the effects of new treatments.
The rat is most commonly used for the 6-OHDA PD model due to established stereotactic techniques and reasonable cost. Typically, only one hemisphere is injected, introducing a unilateral lesion leading to asymmetric motor behavior. The asymmetric motor behaviors, such as circling behavior, result from a physiological imbalance between the lesioned and intact sides. About 1 week after injection, the dopamine pools on the lesioned side are depleted and lead to up-regulation and hypersensitivity of the dopamine receptors on the lesioned side. When the animals are challenged with apomorphine, a dopamine receptor agonist, the stimulation of these up-regulated receptors leads to contralateral circling (away from the lesioned side). The circling behavior can be quantified and used to access the efficacy of potential PD therapeutic agents. In addition, presence of the dopamine-producing neurons in the brain tissue can be identified by immunohistochemistry and Western blot of tyrosine hydroxylase in the brain tissues, and by in situ hybridization of the tyrosine hydroxylase mRNA in the neurons. Furthermore, detection of apoptotic dopaminergic cells can be carried out by immunohistochemistry and Western blot of caspase 3 in the brain tissues, and by in situ hybridization of the caspase 3 mRNA in the neurons. The techniques of immunohistochemistry, Western blot, and in situ hybridization are well known in the art.
Currently, the most widely used treatment for Parkinsonism is administration of L-DOPA, a precursor of dopamine which acts indirectly by replacing the missing dopamine. However, disadvantages are associated with the use of L-DOPA, for example, patients often suffer from side-effects such as dyskinesia and on-off effects, and it is necessary to administer L-DOPA in conjunction with a peripheral dopa-decarboxylase inhibitor such as carbidopa or benzaseride. These inhibitors prevent the peripheral degradation of levodopa to dopamine, thus enabling more drug to enter the brain and limiting peripheral side-effects. Such treatment improves quality of life for patients but does not halt disease progression. Furthermore, such treatment is associated with a number of adverse effects including nausea, vomiting, abdominal distension and psychiatric side-effects (for example, toxic confusional states, paranoia and hallucinations).
An alternative form of therapy is to administer postsynaptic dopamine agonists, for example ergot alkaloids such as bromocriptine—however, this approach is also associated with side-effects. For example, patients receiving bromocriptine often experience dyskinesia psychiatric problems, and in a small number of cases experience vasopastic phenomena and angina. Bromocriptine also causes psychiatric side-effects such as hallucinations. Thus, there is a continuing need for finding an effective and safe medicaments to treat patients with Parkinson disease.
In the invention to be presented in the following sections, a novel use of sporoderm-broken germination-activated Ganoderma spores (GASP) from Ganoderma lucidum as an effective, safe and practical alternative to prevent or treat Parkinson's Disease is described. The GASP has previously been disclosed for use in treating patients with cancer, AIDS, hepatitis, diabetes, cardiovascular diseases and spinal cord injury, and can prevent or inhibit free radical oxidation and hepatotoxic effects. See U.S. Pat. Nos. 6,316,002 and 6,468,542, which are incorporated herein by reference. To date, however, there have been no reports on the effects of GASP on Parkinson's Disease. A further benefit of using the GASP is that they are non-toxic so that higher dosage can be prescribed to the patients.